Murine Intestinal Crypts Research Articles

Synthetic Photoresponsive Hydrogels Enable In Situ Control Over Murine Intestinal Monolayer Differentiation and Crypt Formation

AbstractAs a model of the intestinal epithelium, intestinal stem cells (ISCs) are grown and differentiated as monolayers on materials where stochastic organization of the crypt and villi cells occurs. An allyl sulfide crosslinked photoresponsive hydrogel with a shear modulus of 1.6 kPa is developed and functionalized with GFOGER, Bm‐binder peptide ligands for monolayer growth of ISCs. The allyl sulfide chemistry allows in situ control of mechanics in the presence of growing ISC monolayers and structured irradiation affords spatial regulation of the hydrogel properties. Specifically, ISC monolayers grown on 1.6 kPa substrates are in situ softened to 0.29 kPa, using circular patterns 50, 75, and 100 µm in diameter, during differentiation, resulting in control over the size and arrangement of de novo crypts and monolayer cellularity. These photoresponsive materials should be useful in applications ranging from studying crypt evolution to drug screening and transport across tissues of changing cellular composition. Spatiotemporal softening enables control over the size and arrangement of de novo crypts within intestinal monolayers.

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model

BackgroundMicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage.ResultsWe leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development.ConclusionThe present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

Length limitation of astral microtubules orients cell divisions in murine intestinal crypts

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

Generating and Utilizing Murine Cas9-Expressing Intestinal Organoids for Large-Scale Knockout Genetic Screening.

Organoid culture faithfully reproduces the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumorigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesis-based screening method using murine intestinal organoids by investigating the phenotypical morphology of Cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their untransduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via phalloidin and propidium iodide (PI) staining, proliferation assay and qRT-PCR and also biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis, and drug screening, thereby reducing and potentially replacing animal models.

IFN-γ and IL-17A regulate intestinal crypt production of CXCL10 in the healthy and inflamed colon.

During intestinal inflammation, immature cells within the intestinal crypt are called upon to replenish lost epithelial cell populations, promote tissue regeneration, and restore barrier integrity. Inflammatory mediators including TH1/TH17-associated cytokines influence tissue health and regenerative processes, yet how these cytokines directly influence the colon crypt epithelium and whether the crypt remains responsive to these cytokines during active damage and repair, remain unclear. Here, using laser-capture microdissection and primary colon organoid culture, we show that the cytokine milieu regulates the ability of the colonic crypt epithelium to participate in proinflammatory signaling. IFN-γ induces the TH1-recruiting, proinflammatory chemokine CXCL10/IP10 in primary murine intestinal crypt epithelium. CXCL10 was also induced in colonic organoids derived from mice with active, experimentally induced colitis, suggesting that the crypt can actively secrete CXCL10 in select cytokine environments during colitis. Colon expression of cxcl10 further increased during infectious and noninfectious colitis in Il17a-/- mice, demonstrating that IL-17A exerts a negative effect on CXCL10 in vivo. Furthermore, IL-17A directly antagonized CXCL10 production in ex vivo organoid cultures derived from healthy murine colons. Interestingly, direct antagonism of CXCL10 was not observed in organoids derived from colitic mouse colons bearing active lesions. These data, highlighting the complex interplay between the cytokine milieu and crypt epithelia, demonstrate proinflammatory chemokines can be induced within the colonic crypt and suggest the crypt remains responsive to cytokine modulation during inflammation.NEW & NOTEWORTHY Upon damage, the intestinal epithelium regenerates to restore barrier function. Here we observe that the local colonic cytokine milieu controls the production of procolitic chemokines within the crypt base and colon crypts remain responsive to cytokines during inflammation. IFN-γ promotes, while IL-17 antagonizes, CXCL10 production in healthy colonic crypts, while responses to cytokines differ in inflamed colon epithelium. These data reveal novel insight into colon crypt responses and inflammation-relevant alterations in signaling.

The double stranded RNA analog poly-IC elicits both robust IFN-λ production and oncolytic activity in human gastrointestinal cancer cells

PurposeType III IFN (IFN-λ) is the dominant frontline response over type I IFN in human normal intestinal epithelial cells upon viral infection, this response being mimicked by the dsRNA analog poly-IC. Poly-IC also induces cell death in murine intestinal crypts ex vivo. Here we examined whether these innate defense functions of normal intestinal epithelial cells are recapitulated in gastrointestinal carcinoma cells so that they could be harnessed to exert both immunoadjuvant and oncolytic functions, an unknown issue yet.Experimental designFour human gastrointestinal carcinoma cell lines versus the Jurkat lymphoma cell line were used to assess the effects of intracellular poly-IC on i) IFN-λ secretion and cell proliferation and ii) role of NFκB signaling using the NFκB inhibitory peptide SN50 as a screening probe and a siRNA approach.ResultsPoly-IC induced in all cell lines except Jurkat both a robust IFN-λ secretion and a cytoreductive effect on adherent cells, restricted to proliferating cells and associated with cellular shedding and reduced clonogenicity of the shed cells. Collectively these findings demonstrate the oncolytic activity of poly-IC. Inhibiting NFκB in T84 cells using a siRNA approach decreased IFN-λ production without protecting the cells from the poly-IC oncolytic effects. In line with these findings IFN-λ, that upregulated the anti-viral protein MxA, was unable per se to alter T84 cell proliferation.ConclusionOur demonstration that poly-IC-induced concomitant recapitulation of two innate functions of normal intestine, i.e. IFN-λ production and cell death, by human gastrointestinal cancer cells opens new perspectives in gastrointestinal cancer treatment.

The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress-Driven Mucus Depletion and Exacerbates Intestinal Inflammation.

Background & AimsFood additives, such as emulsifiers, stabilizers, or bulking agents, are present in the Western diet and their consumption is increasing. However, little is known about their potential effects on intestinal homeostasis. In this study we examined the effect of some of these food additives on gut inflammation.MethodsMice were given drinking water containing maltodextrin (MDX), propylene glycol, or animal gelatin, and then challenged with dextran sulfate sodium or indomethacin. In parallel, mice fed a MDX-enriched diet were given the endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA). Transcriptomic analysis, real-time polymerase chain reaction, mucin-2 expression, phosphorylated p38 mitogen-activated protein (MAP) kinase quantification, and H&E staining was performed on colonic tissues. Mucosa-associated microbiota composition was characterized by 16S ribosomal RNA sequencing. For the in vitro experiments, murine intestinal crypts and the human mucus-secreting HT29-methotrexate treated cell line were stimulated with MDX in the presence or absence of TUDCA or a p38 MAP kinase inhibitor.ResultsDiets enriched in MDX, but not propylene glycol or animal gelatin, exacerbated intestinal inflammation in both models. Analysis of the mechanisms underlying the detrimental effect of MDX showed up-regulation of inositol requiring protein 1β, a sensor of ER stress, in goblet cells, and a reduction of mucin-2 expression with no significant change in mucosa-associated microbiota. Stimulation of murine intestinal crypts and HT29-methotrexate treated cell line cells with MDX induced inositol requiring protein 1β via a p38 MAP kinase–dependent mechanism. Treatment of mice with TUDCA prevented mucin-2 depletion and attenuated colitis in MDX-fed mice.ConclusionsMDX increases ER stress in gut epithelial cells with the downstream effect of reducing mucus production and enhancing colitis susceptibility.

Wnt/PCP controls spreading of Wnt/β-catenin signals by cytonemes in vertebrates.

Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

Mammalian small intestinal (SI) tight junctions (TJ) link epithelial cells to one another and function as aperm selective barrier, strictly modulating the passage of ions and macromolecules through the “pore” and “leak” pathways, respectively, thereby preventing absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. SI epithelial permeability is ascribed primarily to properties of TJ between adjoiningenterocytes (ENT), because there is almost no information on TJ composition and paracellular permeability of non‐enterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. In the current study we directed murine intestinal crypts to form specialized organoids highly enrichedin stem cells (ISC), absorptive ENTs, secretory goblet (GOB) or Paneth cells(PAN). Morphological and morphometriccharacteristics of these cells in organoids were similar to those in vivo. Expression of certain TJ genes varied with cell type. Occludin and tricellulin levels were high in both ISC and PAN organoids, while claudin‐1, junctional adhesion molecule‐1, and cingulin were greatest mainly in PAN. Claudin‐2 was greatest in ISC and claudin‐7 expression was greatest in ENT organoids. In contrast, claudin‐15, zonula occludens 1 (ZO‐1) and e‐cadherin distribution were relatively homogeneous among cell types. Immunofluorescent staining showed that e‐cadherin and claudin‐7 marked mainly the basolateral membrane, while claudin‐2, ZO‐1 and occludin resided apically. Remarkably, organoids enrichedin ENTs and GOBs, respectively were 3.7‐fold and 3.4‐fold more permeable to 4 kda dextran thanthose enriched in ISCs (P<0.05). Similarly, ENT and GOB were 2.9 and2.6‐fold more permeable to 10 kda dextran compared to ISC organoids. The trend was similar (>2‐fold)for ENT and GOB vs. PAN organoids for both 4 and 10 kda dextran flux (P<0.05). As was expected, for all cell types 10 kda dextran flux was significantly lower than 4 kda (P < 0.05). A time course of dextran flux in ENT and PAN organoids revealed that 4 kda dextran entered the lumen of ENT by 10 min and highest intensity was observed by 30 min (P<0.05). In contrast, dextran flux was nearly absent in PAN organoids, regardless of time point. Additionally, the TJ regulator larazotide prevented the ~10‐fold increases in dextran flux induced by the TJ disrupter AT1002 in both ISC and ENT organoids (P < 0.05), indicating that AT1002 non‐selectively increases permeability. Lastly, dedifferentiation of mature ENTs results inreacquisition of ISC‐like characteristics in TJ composition and dextranpermeability, suggesting that post‐differentiation properties of TJs are not hard wired. In conclusion, differentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellularpathway of epithelia.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 (RPF) and 1456673 (RPF) and by NIH Grant R01‐DK‐102934 (NG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

BackgroundMammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia.ResultsHere we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired.ConclusionsDifferentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Separation of Dual Oxidase 2 and Lactoperoxidase Expression in Intestinal Crypts and Species Differences May Limit Hydrogen Peroxide Scavenging During Mucosal Healing in Mice and Humans.

DUOX2 and DUOXA2 form the predominant H2O2-producing system in human colorectal mucosa. Inflammation, hypoxia, and 5-aminosalicylic acid increase H2O2 production, supporting innate defense and mucosal healing. Thiocyanate reacts with H2O2 in the presence of lactoperoxidase (LPO) to form hypothiocyanate (OSCN-), which acts as a biocide and H2O2 scavenging system to reduce damage during inflammation. We aimed to discover the organization of Duox2, Duoxa2, and Lpo expression in colonic crypts of Lieberkühn (intestinal glands) of mice and how distributions respond to dextran sodium sulfate (DSS)-induced colitis and subsequent mucosal regeneration. We studied tissue from DSS-exposed mice and human biopsies using in situ hybridization, reverse transcription quantitative polymerase chain reaction, and cDNA microarray analysis. Duox2 mRNA expression was mostly in the upper crypt quintile while Duoxa2 was more apically focused. Most Lpo mRNA was in the basal quintile, where stem cells reside. Duox2 and Duoxa2 mRNA were increased during the induction and resolution of DSS colitis, while Lpo expression did not increase during the acute phase. Patterns of Lpo expression differed from Duox2 in normal, inflamed, and regenerative mouse crypts (P < 0.001). We found no evidence of LPO expression in the human gut. The spatial and temporal separation of H2O2-consuming and -producing enzymes enables a thiocyanate- H2O2 "scavenging" system in murine intestinal crypts to protect the stem/proliferative zones from DNA damage, while still supporting higher H2O2 concentrations apically to aid mucosal healing. The absence of LPO expression in the human gut suggests an alternative mechanism or less protection from DNA damage during H2O2-driven mucosal healing.

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts

BackgroundA restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities.ResultsTwo taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems.ConclusionsOur comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

12-O-tetradecanoylphorbol-13-acetate (TPA) increases murine intestinal crypt stem cell survival following radiation injury.

Radiation enteropathy is a common complication in cancer patients following radiation therapy. Thus, there is a need for agents that can protect the intestinal epithelium against radiation. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce differentiation and/or apoptosis in multiple cell lines and primary cells. In the current report, we studied the function of TPA in radiation induced enteropathy in cultured rat intestinal epithelial cell line IEC-6 after ionizing radiation (IR) and in mice after high dose total-body gamma-IR (TBI). In IEC-6 cells, there were reduced apoptosis and cell cycle arrest in TPA treated cells after IR. We detected a four-fold increase in crypt cell survival and a two-fold increase in animal survival post TBI in TPA treated mice. The beneficial effects of TPA were accompanied by upregulation of stem cells markers and higher level of proteins that are involved in PKC signaling pathway. In addition, TPA also decreased the TBI-augmented levels of the DNA damage indicators. The effects were only observed when TPA was given before irradiation. These results suggest that TPA has the ability to modulate intestinal crypt stem cells survival and this may represent a promising countermeasure against radiation induced enteropathy.

Reg4 + deep crypt secretory cells function as epithelial niche for Lgr5 + stem cells in colon

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.

Organoids as an ex vivo model for studying the serotonin system in the murine small intestine and colon epithelium

Intestinal organoids were recently established as an ex vivo model of the intestinal epithelium. The present study investigated the serotonin (5-hydroxytryptamine, 5-HT) system using organoids. Organoids from murine small intestinal and colonic crypts were successfully cultured. Reverse transcription–polymerase chain reaction (RT-PCR) analysis showed that small intestinal and colonic organoids express mRNAs encoding tryptophan hydroxylase-1 (TPH1) (the rate-limiting enzyme of 5-HT synthesis), serotonin reuptake transporter (SERT), 5-HT receptor (HTR)2A, HTR2B, and HTR4. SERT mRNA levels were significantly higher in the small intestine than in the colon in both the mucosal tissues and organoids, as estimated by quantitative real-time RT-PCR. Although the 5-HT concentration and levels of chromogranin A (CgA) (an enteroendocrine cell marker), TPH1, and HTR4 mRNAs were significantly higher in the colonic mucosa than the small intestinal mucosa, they were the same in small intestinal and colonic organoids. There were no significant differences in HTR2A and HTR2B mRNA levels between the small intestine and colon in either the mucosal tissues or organoids. Immunofluorescence staining showed that the number of CgA-positive cells in the colonic organoids appeared to increase upon culturing with acetate. Acetate supplementation significantly increased CgA, TPH1, and HTR4 mRNA levels in the colonic organoids. We propose that organoids are useful for investigating the 5-HT system in the intestinal epithelium, even though colonic organoids may require gut microbiota–derived factors such as short-chain fatty acids.

Analysis of Cell Death Induction in Intestinal Organoids In Vitro.

The intestinal epithelium has an important function in the absorption of nutrients contained in the food. Furthermore, it also has an important barrier function, preventing luminal pathogens from entering the bloodstream. This single cell layer epithelium is quite sensitive to various cell death-promoting triggers, including drugs, irradiation, and TNF family members, leading to loss of barrier integrity, epithelial erosion, inflammation, malabsorption, and diarrhea. In order to assess the intestinal epithelium-damaging potential of treatments and substances specific test systems are required. As intestinal tumor cell lines are a poor substitute for primary intestinal epithelial cells, and in vivo experiments in mice are costly and often unethical, the use of intestinal organoids cultured from intestinal crypts provide an ideal tool to study cell death induction and mechanisms in primary intestinal epithelial cells. This protocol describes the isolation and culture of intestinal organoids from murine small intestinal crypts, and the quantitative assessment of cell death induction in these organoids.

Viral oncogene expression in the stem/progenitor cell compartment of the mouse intestine induces adenomatous polyps.

Genetic and epigenetic events that alter gene expression and/or protein function or localization are thought to be the primary mechanism that drives tumorigenesis and governs the clinical behavior of cancers. Yet, a number of studies have shown that the effects of oncogene expression or tumor suppressor ablation are highly dependent on cell type. The molecular basis for this cell-type specificity and how it contributes to tumorigenesis are unknown. Here, expression of a truncated SV40 large T antigen in murine intestinal crypts promoted the formation of numerous adenomatous polyps in the colon and small intestine. In contrast, when the same T-antigen construct is expressed in villous enterocytes, the consequences are limited to hyperplasia and dysplasia. The T-antigen-induced polyps show high levels of the proto-oncogene c-Myc protein even though there is no transport of β-catenin to the nucleus. Targeting the expression of viral oncogenes to intestinal crypts or villi provides a murine model system for studying cell-type specific effects in tumorigenesis, and is particularly relevant to the study of APC/β-catenin-independent pathways contributing to the generation of intestinal polyps. This mouse model system describes the formation of colon polyps in the absence of Wnt/β-catenin signaling.